Progesterone (P) stimulates prolactin secretion through an unknown neural mechanism in estrogen (E) primed female monkeys. Serotonin also stimulates prolactin secretion and this laboratory demonstrated that E induces nuclear progestin receptors (PR) in serotonin neurons. Thus, PR in serotonin neurons could transduce the action of P on prolactin secretion. Studies were performed to determine (1) whether blocking nuclear PR would block P-induced prolactin secretion and, conversely, (2) whether increasing serotonin concentrations in the synapse would augment P-induced prolactin secretion. In both studies, female monkeys were spayed, adapted to a vest and tether remote sampling system and catheterized prior to experiments. To block nuclear PR and not membrane PR, RU486 (2mg/kg) or ethanol vehicle (control)was administered with a P injection. To increase serotonin in the synapse, the serotonin reuptake inhibitor, fluoxetine (5mg/day) was infused for 4 weeks. P was injected during the week of vehicle infusion and during the last week of fluoxetine infusion. RU486 completely blocked the P-induced prolactin surge. In addition, fluoxetine significantly increased prolactin secretion during the P-induced prolactin peak compared to equal time points during saline infusion. These data indicate that P induces prolactin via a genomic mechanism and not through a membrane action. The data also support a pivotal role for serotonin in the neural regulation of P-induced prolactin secretion.